ANTIBODY THERAPEUTICS

INTRODUCTION

Teneobio has developed unique technologies, including a

transgenic rat platform, UniRat®, expressing human heavy chain

antibodies (UniAbsTM) and a state-of-the-art sequence-based dis-

covery engine to create novel multispecific antibodies for various

therapeutic indications. In addition to therapeutic antibodies,

UniAb binding domains can be successfully used as 1) antigen

recognition domains on CAR T-cells, 2) targeting moieties for

nanoparticles, 3) antibody drug-, toxin- or radiolabel-conjugates,

and 4) viral payloads (eg, to modify the tumor microenvironment).

Using this unique technology, Teneobio has identified an unprece-

dented number of novel anti-CD3s, which in the context of bi- or

multispecifics, enable maximal T-cell redirection for tumor cytotox-

icity and minimal cytokine release. Teneobio’s unique capabilities

and anti-CD3 platform are being applied to develop a number

of breakthrough multispecific therapeutic candidates to treat

hematological and solid tumor cancers. Its lead mutispecific ther-

apeutic program, anti-BCMAxCD3, is in preclinical development

for a planned IND submission in 2018.

ANTIBODY DISCOVERY

The past 30 years has seen a rapid evolution of therapeutic

antibody technologies. A transformative milestone was the gen-

eration of transgenic human Ig rodent platforms, which enable

the discovery of fully human antibodies with considerably less im-

munogenicity, overcoming the need to chimerize, humanize, and

affinity mature mouse antibodies.1 A survey of more than 50 cur-

rently marketed antibody therapeutics further highlights the fidelity

and success of rodent-derived antibodies compared to in vitro dis-

play approaches to antibody discovery.2 Not surprisingly, in vivo-

derived antibodies have advantages conferred by physiological

selection for critical quality attributes, including stability, solubility,

and high affinity. In contrast, in vitro display methods lack these

advantages. Hence, transgenic human Ig rodents have become

a mainstay of therapeutic antibody discovery in the biopharma-

ceutical industry.

In the 90s, the engineering of antibodies extended beyond

humanization and affinity maturation technologies to include the

rational design of antibody Fcs through the selection of isotypes,

the engineering for enhanced or silenced immune effector func-

tions and the design for extended or reduced half-life.3 The gen-

eration and engineering of antibodies (eg, scFvs, llama VHHs,

human VHs) or alternative scaffolds (eg, DARPins, Centyrins,

Fynomers) further afforded the ability to generate bi- and multi-

specific therapeutic candidates through the assembly of modular

domains that were linked chemically or by amino acids.4-6 Addi-

tional technologies involved the “knobs-into-holes” Fc heterodimer-

ization and variations thereof, to enable bispecific generation in

the natural and structurally conserved antibody format.6 Through

the years, an explosion of multispecific formats ensued, some of

which progressed to clinical trials while others failed in develop-

ment from challenges in manufacturability or limiting biology.

With this backdrop, the next generation of transformational anti-

body therapeutics will reach beyond the monospecific, bivalent

format toward physiologically compatible and developable

human multispecific antibodies with improved or de novo biology,

overcoming the therapeutic limitations of native human IgGs.

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Teneobio’s Next Generation of Multispecific Antibody Therapeutics By: Omid Vafa, PhD, MBA

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Advances in molecular and high-

throughput technologies are enabling inno-

vative approaches to discovering and

capturing antibody diversity, previously

limited by clonal loss in traditional hy-

bridoma generation. Specifically, next-gen-

eration sequencing has enabled

comprehensive profiling of full antibody

repertoires of immunized organisms. Fur-

thermore, using advanced methods of

gene assembly, one can synthesize thou-

sands of unique antibody sequences to be

expressed and screened in high-throughput

format. Taken together, these technologies

enable rapid screening and identification

of affinity-matured functional antibody

leads at unprecedented speeds. Using nat-

urally derived human antibodies from

transgenic rats and state-of-the-art se-

quence-based antibody discovery, Teneo-

bio is developing the next generation of

novel and manufacturable multispecific an-

tibodies as therapeutics for oncology, im-

munology, and infectious diseases.

DISCOVERY PLATFORMS:UNIRAT & TENEOSEEK

Teneobio’s human Ig transgenic plat-

form, the UniRat, is based on a triple

knockout rat wherein the expressions of the

native variable coding sequences and the

heavy and light chain constant regions

have been inactivated. The UniRat has

been genetically modified to exclusively

express the full human VDJ repertoire (all

VH families), with transgenes of human

heavy chain variable domains linked to a

conserved rat Fc. Immunization of the Uni-

Rat elicits a normal antibody response that

results in the expression of UniAbs, human

heavy-chain-only antibodies of approxi-

mately 80 kDa, contrasting with the stan-

dard ~150-kDa human IgG. Importantly,

heavy chain variable domains from the

UniRat, UniDabsTM, are the smallest anti-

gen-binding units of a human IgG at ap-

proximately 12.5 kDa (~100 amino acids)

and can be assembled as modular do-

mains of multispecifics. Figure 1 illustrates

a subset of such multispecific formats, en-

abling the generation of a plenitude of

specificities against different epitopes on

the same antigen or different specificities

for different antigens. Heterodimerization

of such heavy-chain-only multispecifics or

their combination with standard heavy-

light-chain formats is feasible, given that

UniDabs (VH domains) do not interact with

either kappa or lambda light chains in

vitro or when co-expressed in cell lines.

Complementing Teneobio’s UniRat

platform is a proprietary next-generation

sequence-based discovery engine called

TeneoSeek. The TeneoSeek discovery en-

F I G U R E 1

“The advents of immune-oncology checkpoint inhibitors and multispecific antibodytechnologies enable the redirection of the immune system for targeted killing of can-cers of interest. The past decade has seen an exponential increase in such therapeu-tics, including CAR T-cell therapy, directed nanoparticles delivering payloads,antibody drug-, toxin-, and radiolabel-conjugates, etc. Teneobio’s UniRat andTeneoSeek platforms, combined with a tool kit of engineering capabilities afford theopportunity to rapidly and effectively identify antibody therapeutic leads as well asUniDabs for a variety of multispecifics and novel cellular and delivery technologies.”

UniRat®-derived multispecifics with flexible design and tune-able affinity and avidity.

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gine comprises three key components: 1)

next-generation sequencing of the full

repertoire of the immunized UniRat, 2)

bioinformatic B-cell lineage analysis of

human variable domain sequences to iden-

tify antigen-specific CDR families, and 3)

high throughput gene assembly, expres-

sion and functional screens to identify

leads of interest. Inherent in the bioinfor-

matic analysis are the proprietary algo-

rithms that eliminate variable domain

sequences of CDRs containing post-trans-

lational liabilities (eg, cysteines, methion-

ines, deamidation motifs, glycosylation

residues, etc) as well as problematic sur-

face patch residues that can contribute to

instability and aggregation. The start-to-fin-

ish timeline, from immunization to lead

identification of UniAbs, is approximately

4 months, one of the fastest in the industry

(Figure 2).

The rich diversity of antibody leads

obtained from UniRat immunizations, cou-

pled with bioinformatic analysis of se-

quences and lineage afford the ability to

identify specific UniAb members (from VH

CDR3 families) against a broad number of

epitopes and a wide range of affinities

spanning pM to µM. Uniquely, UniDabs

have been shown to access protein

grooves and crevices on target antigens

that are otherwise non-accessible with stan-

dard H2L2 antibodies, given the near dou-

ble size of their paratopes. Modular

UniDabs can then be rapidly assembled

and further engineered for desired multi-

specificity and effector functions on human

Fc backbones of interest. Of note, the

highly manufacturable and stable UniDabs

and their derived multispecifics, which

have a melting temperature of ~60°C to

64°C, can be expressed at grams per liter

and are easily manufactured in CHO cell

lines. CHO cell supernatant yields of het-

erodimeric UniAb multispecifics are > 85%

and can be purified using a single capture

step process to 98% purity. The developa-

bility and expression profiles of UniAbs

and their multispecific antibody derivatives

are quite similar to that of standard anti-

bodies and compatible with industry stan-

dard manufacturing platforms.

UNIQUE T-CELL REDIRECTIONPLATFORM FOR CANCERTHERAPY

Throughout the past decade, T-cell

redirection using bispecific antibodies has

provided favorable clinical outcomes in

treatments of liquid tumors, including

leukemia and lymphoma. Teneobio has

used its technologies to provide unique so-

lutions for T-cell redirecting therapies. The

basis for the approach relies on the cou-

pling of an anti-CD3 recognition domain

with a targeting moiety as a fusion con-

struct. In the past, a limitation to this ap-

proach has been partly target-related,

given the difficulty of generating anti-CD3

antibodies, as well as CD3 biology.

Specifically, limitations associated with the

immunogenicity of CD3 epitopes and the

industry’s modification and/or humaniza-

tion of less than a handful of known anti-

CD3 antibodies (eg, OKT3 and SP34)

have limited the application of this power-

ful approach to cancer therapy.7 To ad-

dress these limitations, the TeneoSeek

discovery platform was applied to identify

> 100 unique anti-CD3 sequences span-

ning different target epitopes and covering

a broad spectrum of affinities from low nM

to µM affinities. Largely enabled by se-

quence and repertoire lineage analysis,

leads from these efforts have yielded a di-

verse collection of novel anti-CD3 antibod-

ies with unique and differentiated biology.

Recent clinical utility and the therapeu-

tic application of bispecific anti-CD3 anti-

bodies have been complicated by adverse

events, including cytokine release syn-

drome and neurotoxicity. In contrast to

these first-generation anti-CD3 molecules,

Teneobio’s unique anti-CD3 platform is

based on the access to a diverse set of

anti-CD3s with different binding and T-cell

activation profiles, decoupling tumor-spe-

cific cytotoxicity from cytokine release. A

comprehensive analysis of a subset of

these leads has yielded variants that can

differentially kill cancer cells with minimal

proinflammatory cytokine release, poten-

tially increasing the therapeutic window.

This would be particularly advantageous

for anti-CD3 bispecifics with longer in vivo

half-lives. The ability to tailor anti-CD3-

based bispecifics for targets of interest of-

fers prospects of a next generation of safer

and improved therapies using clinically

validated cancer cell targets. These safetyDrug

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F I G U R E 2

The TeneoSeek discovery engine.

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profiles are currently being assessed in

vivo, and studies to date indicate that mul-

tispecifics derived from the UniAb platform

exhibit mouse and monkey half-lives that

are consistent with that of standard anti-

bodies. Additional in vivo studies have val-

idated the anti-CD3 platform,

demonstrating the efficacy of the bis-

pecifics in weekly dosing of mouse mod-

els. With a planned IND filing for 2018,

the clinical validation of Teneobio’s anti-

CD3 platform and lead therapeutic candi-

date, anti-BCMAxCD3 (bivalent for

BCMA, monovalent for CD3) for the treat-

ment of multiple myeloma, may open new

opportunities to address challenges related

to efficacy and adverse events associated

with T-cell targeting of liquid and solid tu-

mors. To this end, Teneobio is applying its

T-cell redirection platform in additional

therapeutic discovery programs, including

anti-CD22xCD3 and anti-CD19xCD22xCD3

for lymphoma and ALL as well as anti-PS-

MAxPSCAxCD3 for prostate cancer.

NEXT-GENERATIONMULTISPECIFIC UNIAB-BASEDTHERAPEUTICS

The advents of immune-oncology

checkpoint inhibitors and multispecific an-

tibody technologies enable the redirection

of the immune system for targeted killing of

cancers of interest. The past decade has

seen an exponential increase in such ther-

apeutics, including CAR T-cell therapy, di-

rected nanoparticles delivering payloads,

antibody drug-, toxin-, and radiolabel-con-

jugates, etc. Teneobio’s UniRat and

TeneoSeek platforms, combined with a tool

kit of engineering capabilities afford the

opportunity to rapidly and effectively iden-

tify antibody therapeutic leads as well as

UniDabs for a variety of multispecifics and

novel cellular and delivery technologies.

The ease of assembling multispecifics with

modular binding domains enables the ex-

ploration of synergies to activate and redi-

rect the immune system to cancer cells.

Multispecific UniAbs and UniDabs can be

assembled for improved affinity through in-

creased avidity against targets of interest

(eg, a bivalent heavy chain only anti-

BCMA can competitively block APRIL lig-

and binding to BCMA). Additionally,

UniAbs can be optimized for tissue speci-

ficity and selectivity through avidity for dif-

ferent antigens co-expressed on tissue

targets of interest. Bi- or multiparatopic mul-

tispecifics can elicit gain-of-function or de

novo activities, otherwise absent in mono-

specific antibodies or with their combina-

tions. Biological activation or redirection

of T-cells can be further explored to assess

multispecific combinations that offer the

best efficacy and safety profiles. More-

over, the application of UniDabs as extra-

cellular domains for CAR T-cells has been

validated and shown to be superior to the

use of scFvs in some settings. The applica-

tions of UniDabs as antibody-drug/radio-

label conjugates, targeting moieties on

nanoparticles, viral payloads to modify the

tumor microenvironment, or imaging tools

(given their relatively small size and tumor

penetrance) offer endless possibilities to

exploit these human variable domains for

therapeutic benefit. Teneobio’s unique plat-

forms and antibody drug discovery capa-

bilities are poised to deliver on these goals

for a variety of indications, including on-

cology, immune disorders, and infectious

diseases. ◆

REFERENCES

1. Lonberg, N. Fully human antibodies from transgenic mouse and phagedisplay platforms. Curr Opin Immunol. 2008 Aug;20(4):450-9.

2. https://en.wikipedia.org/wiki/List_of_therapeutic_monoclonal_antib-odies

3. Gilliland GL, Luo J, Vafa O, Almagro JC. Leveraging SBDD in proteintherapeutic development: antibody engineering. Methods Mol Biol.2012;841:321-49.

4. Muyldermans S1, Cambillau C, Wyns L. Recognition of antigens bysingle-domain antibody fragments: the superfluous luxury of paired do-mains. Trends Biochem Sci. 2001 Apr;26(4):230-5.

5. Vazquez-Lombardi R, Phan TG, Zimmermann C2, Lowe D, Jermutus L,Christ D.Challenges and opportunities for non-antibody scaffold drugs.Drug Discov Today. 2015 Oct;20(10):1271-83.

6. Spiess C, Zhai Q, Carter PJ. Alternative molecular formats and thera-peutic applications for bispecific antibodies. Mol Immunol. 2015Oct;67(2 Pt A):95-106.

7. Salmerón A1, Sánchez-Madrid F, Ursa MA, Fresno M, Alarcón B. Aconformational epitope expressed upon association of CD3-epsilonwith either CD3-delta or CD3-gamma is the main target for recognitionby anti-CD3 monoclonal antibodies. J Immunol. 1991 Nov1;147(9):3047-52.

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B I O G R A P H Y

Dr. Omid Vafa is the Chief BusinessOfficer at Teneobio, Inc. He has more than15 years of global business and scientificexperience in biotechnology andbiopharmaceutical drug discovery, assetevaluation, transactions, and licensing.Prior to joining Teneobio, he was theDirector of Strategy and ScientificPartnerships at Janssen PharmaceuticalCompanies of Johnson and Johnson andthe Biotechnology and Oncology Lead atthe London Innovation Center of J&J. Heearned his PhD at Georgetown University,his MBA from the Villanova UniversitySchool of Business, and his BS from theUniversity of California at Irvine. Hecompleted his post-doctoral fellowships atThe Scripps Research Institute (TSRI) and theSalk Institute for Biological Studies in LaJolla, California. He can be contacted atovafa@teneobio.com.

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